| Abstract
| - Inhibition of urokinase activity represents a promising target for antimetastatic therapy for severaltypes of tumor. The present study sets out to investigate the potential of Raman spectroscopy for defining themolecular details of inhibitor binding to this enzyme, with emphasis on single crystal studies. It is demonstratedthat high quality Raman spectra from a series of five inhibitors bound individually to the active site of humanurokinase can be obtained in situ from urokinase single crystals in hanging drops by using a Raman microscope.After recording the spectrum of the free crystal, a solution of inhibitor containing an amidine functional groupon a naphthalene ring was added, and the spectrum of the crystal−inhibitor complex was obtained. The resultingdifference Raman spectrum contained only vibrational modes due to bound inhibitor, originating from theprotonated group, i.e., the amidinium moiety, as well as naphthalene ring modes and features from otherfunctionalities that made up each inhibitor. The identification of the amidinium modes was placed on aquantitative basis by experimental and theoretical work on naphthamidine compounds. For the protonated group,−C−(NH2)2+, the symmetric stretch occurs near 1520 cm-1, and a less intense antisymmetric mode appears inthe Raman spectra near 1680 cm-1. The presence of vibrational modes near 1520 cm-1 in each of the Ramandifference spectra of the five complexes examined unambiguously identifies the protonated form of the amidiniumgroup in the active site. Several advantages were found for single crystal experiments over solution studies ofinhibitor−enzyme complexes, and these are discussed. The use of single crystals permits competitive bindingexperiments that cannot be undertaken in solution in any kind of homogeneous assay format. The Ramandifference spectrum for a single crystal that had been exposed to equimolar amounts of all five inhibitors inthe hanging drop showed only the Raman signature of the compound with the lowest Ki . These findingssuggest that the Raman approach may offer a route in the screening of compounds in drug design applicationsas well as an adjunct to crystallographic analysis.
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